Tissue specific prodrugs

ABSTRACT

The invention provides novel peptide prodrugs which contain cleavage sites specifically cleaved by prostate specific membrane antigen (PSMA). These prodrugs are useful for substantially inhibiting the non-specific toxicity of a variety of therapeutic drugs. PSMA is secreted by prostatic glandular cells. Upon cleavage of the prodrug by PSMA, the therapeutic drug are activated and exert their toxicity. Sesquiterpene-γ-lactones form part of the prodrugs, and are designed to be linked to carrier moieties such as the peptides of the invention. Methods for treating cell proliferative disorders are also featured in the invention.

TECHNICAL FIELD

[0001] This invention relates generally to the targeted activation anddelivery of therapeutic drugs to cells that produce prostate specificmembrane antigen (PSMA) and relates more specifically to PSMA-specificpeptide prodrugs that become activated to yield therapeutic drugs.

BACKGROUND

[0002] There is currently no effective therapy for men with metastaticprostate cancer who relapse after androgen ablation, even thoughnumerous agents have been tested over the past thirty years. Prolongedadministration of effective concentrations of standard chemotherapeuticagents is usually not possible because of dose-limiting systemictoxicities.

[0003] PSMA is a 100 kDa prostate epithelial cell type II transmembraneglycoprotein that was originally isolated from a cDNA library from theandrogen responsive LNCaP human prostate cancer cell line, as disclosed,for example by Horoszewicz et al., Cancer Res. 43:1809-1818, (1980).Immunohistochemical studies using monoclonal antibodies havedemonstrated that PSMA is expressed by normal prostate epithelium and iseven more highly expressed by a large proportion of prostate cancers,including metastatic prostate cancers, as disclosed, for example inHoroszewicz et al.; Wright et al., Urol. Oncol 1:18-28, (1995); andLopes et al., Cancer Res. 50:6423-6429, (1990). Low-level detection ofthe PSMA protein has also been seen in the duodenal mucosa and in asubset of proximal renal tubules. In all other human tissues, includingnormal vascular endothelium, PSMA expression was not detectable, asdisclosed for example, in Silver et al., Clin. Cancer Res. 3:81-85,(1997); and Chang et al., Cancer Res. 59:3192-3198, (1999). PSMA,however, has been detected in the neovasculature of a large number ofdifferent tumor types including breast, renal, colon, pancreatic, brain,melanoma, lung, testicular, sarcoma and transitional cell carcinomas(Silver et al., and Chang et al.).

[0004] Two discrete enzymatic functions for PSMA have been described.Carter et al., Proc. Natl. Acad. Sci., USA 93:749-753, (1996),demonstrated that PSMA possesses the hydrolytic properties of anN-acetylated α-linked acidic dipeptidase (NAALADase). NAALADase is amembrane hydrolase activity that is able to hydrolyze the neuropeptideN-acetyl-l-aspartyl-l-glutainate (NAAG) to yield the neurotransmitterglutamate and N-acetyl-aspartate. In addition to the NAALADase activity,PSMA also functions as a pteroyl poly-y-glutamyl carboxypeptidase(folate hydrolase), as disclosed, for example, by Pinto et al., Clin.Cancer Res. 2:1445-1451, (1996). PSMA exhibits exopeptidase activity andhas more recently been classified as glutamate carboxypeptidase II. Itis able to progressively hydrolyze γ-glutamyl linkages of bothpoly-γ-glutamated folates and methotrexate analogs with varying lengthglutamate chains, as disclosed, for example, in Pinto et al., and Hestonet al., Urology 49 (Suppl 3A): 104-112, (1997). PSMA is able toprogressively hydrolyze γ-glutamyl linkages of both poly-gammaglutamated folates and poly-gamma glutamated methotrexate analogs withvarying length glutamate chains. Unfortunately, it has also been foundthat these polyglutamated analogs can also be readily hydrolyzed bygamma glutamyl hydrolase (GGH), a lysosomal enzyme. Gingras et al.recently characterized a human blood plasma glutamate carboxypeptidase(PGCP) that has significant sequence homology to PSMA and glutamatecarboxypeptidase activity, see J. Biol. Chem. 274:11742-11750, (1999).Proteins that are homologous to PSMA have been recently isolated fromthe rat brain and pig jejunum, as disclosed, for example, inLuthi-Carter et al., Proc. Natl. Acad Sci. USA 95:3215-3220, (1998); andHalsted et al., J. Biol. Chem. 273:20417-20424, (1998). These proteinshave >80% amino acid sequence homology with PSMA and possess similarenzymatic functions.

SUMMARY

[0005] The present invention provides therapeutic prodrug compositions,comprising therapeutic drugs linked to peptides, which are efficientlyand specifically cleaved by PSMA. The peptides include amino acidsequences containing cleavage sites for prostate specific membraneantigen (PSMA) and other enzymes with the same overall activity andoverall proteolytic specificity as PSMA. Representative amino acidsequences are provided, and include a-linked dicarboxylic aminoacid-containing peptides, side chain-linked (for example, γ-linked)dicarboxylic amino acid-containing peptides, and α-, side chain-linkeddicarboxylic amino acid-containing peptides, as well as analogs,derivatives and conservative variations thereof. The linkage oftherapeutic drug to peptide substantially inhibits the non-specifictoxicity of the drug. Cleavage of the peptide releases the drug,activating it or restoring its non-specific toxicity.

[0006] Examples of therapeutic drugs include analogs of therapeuticsesquiterpene—lactones, including derivatives of the thapsigargins. Thethapsigargins are a group of natural products isolated from species ofthe umbelliferous genus Thapsia. The term thapsigargins has been definedby Christensen, et al., Prog. Chem. Nat. Prod, 71 (1997) 130-165. Thesederivatives contain a means of linking the therapeutic drug to carriermoieties, including peptides and antibodies, including those peptidesand antibodies which can specifically interact with antigens, includingPSMA. The interactions can involve cleavage of the peptide to releasetherapeutic drugs, for example, sesquiterpene-γ-lactones, such asthapsigargin derivatives.

[0007] The invention also provides a method for treating cellproliferative disorders, including those which involve the production ofPSMA, in subjects having or at risk of having such disorders. The methodinvolves administering to the subject a therapeutically effective amountof the composition of the invention.

[0008] The invention also provides a method of producing the prodrugcomposition of the invention. In another embodiment, the inventionprovides a method of detecting PSMA activity in tissue. In yet anotherembodiment, the invention provides a method of selecting appropriateprodrugs for use in treating cell proliferative disorders involvingPSMA-production.

[0009] The invention also provides a method for detecting a cellproliferative disorder associated with PSMA production in a tissue of asubject, comprising contacting a target cellular component suspected ofhaving a PSMA-associated disorder, with a reagent which detectsenzymatically active PSMA.

[0010] The invention also provides a method of determining PSMA activityin a PSMA-containing sample, comprising contacting the sample with adetectably labeled peptide which is specifically cleaved by PSMA for aperiod of time sufficient to allow PSMA to cleave the peptide, detectingthe detectable label to yield a detection level, which is then comparedto the detection level obtained by contacting the same detectablylabeled peptide with a standard PSMA sample of known activity.

[0011] The invention also provides a method of imaging soft tissueand/or bone metastases which produce PSMA, comprising administering alipophilic imaging label linked to a peptide which is specificallycleaved by PSMA to a subject having or suspected of having aPSMA-associated cell proliferative disorder, allowing PSMA to cleave thepeptide, allowing the lipophilic imaging label to accumulate in thetissue and/or bone, allowing the subject to clear the uncleaved peptide,and imaging the subject for diagnostic purposes.

[0012] As used herein, the term “prostate specific membrane antigen”(PSMA) means prostate specific membrane antigen, as well as all otherproteases that have the same or substantially the same proteolyticcleavage specificity as prostate specific membrane antigen. As usedherein, “sufficiently toxic” refers to therapeutic drugs which displaynonspecific toxicity toward cells with an LC₅₀ concentration (that is,the concentration required to kill 50% of treated cells) that is atleast 3 times lower than the LC₅₀ concentration of the prodrugs of theinvention, more preferably at least 20 times lower, and therapeuticdrugs most preferably have an LC₅₀ concentration that is at least 100times lower than the LC₅₀ concentration of the prodrugs of theinvention. The term “contacting” refers to exposing tissue to thepeptides, therapeutic drugs or prodrugs of the invention so that theycan effectively inhibit cellular processes, or kill cells. Contactingmay be in vitro, for example by adding the peptide, drug, or prodrug toa tissue culture to test for susceptibility of the tissue to thepeptide, drug or prodrug. Contacting may be in vivo, for exampleadministering the peptide, drug or prodrug to a subject with a cellproliferative disorder, such as prostate or breast cancer. By“polypeptide” is meant any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). Amino acids include the 20 common amino acids thatmake up human proteins as well as other unnatural amino acids that maybe substituted for the common amino acids. The term amino acids alsoencompasses the 1- and d-stereoisomers of each amino acid. As writtenherein, amino acid sequences are presented according to the standardconvention, namely that the amino terminus of the peptide is on theleft, and the carboxy terminus on the right.

[0013] Unless otherwise defined, all technical and scientific terms usedherein have the ordinary meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other reference materials mentionedherein are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

[0014] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a structure of an embodiment of a PSMA-activated TGprodrug.

[0016]FIG. 2 is a set of structures for particular embodiments oflinkers which can be linked to amine groups of therapeutic drugs.

[0017]FIG. 3 is a schematic diagram for the synthesis of aspartate- andglutamate-containing thapsigargin analogs, with the synthesis of12-ADT-Glu(8-O-(12-[L-glutamylamino]-dodecanoyl)-8-O-debutanoylthapsigargin)illustrated as an example of the method.

[0018]FIG. 4 is a graph of the percentage inhibition of clonogenicsurvival of TSU cells exposed to various concentrations ofAPA-Glu*Glu*Glu*Glu*Asp in the presence or absence of PSMA.

[0019]FIG. 5 is a scheme of the syntheses of particular thapsigarginanalogs (leucine-containing alkanoyl thapsigargins).

[0020]FIG. 6 is a scheme of the syntheses of particular thapsigarginanalogs (glutamic acid-containing alkanoyl thapsigargins).

[0021] Like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

[0022] PSMA is expressed in high levels by prostate, and other, cancercells but not by normal cells. The specific targeting of the killingability of therapeutic drugs to prostate, and other, cancer cells isenabled. Therapeutic drugs, for example, thapsigargins modified in the8-position, are directly or indirectly coupled to the α-amino, orside-chain carboxyl of a peptide including dicarboxylic acid-containingamino acids or amidated analogs thereof, for example, glutamic acid,aspartic acid, glutamine or asparagine. Linking groups can be bondedbetween the drugs and the peptides.

[0023] The invention involves peptides that contain a cleavage sitespecific for prostate specific membrane antigen (PSMA). These peptidesare efficiently and specifically cleaved by PSMA. These peptides areuseful for substantially inhibiting the non-specific toxicity of thetherapeutic agents prior to the agents coming in proximity to tissuecontaining PSMA. The prodrugs of the invention comprise peptidescontaining a cleavage site specific for PSMA, and therapeutic drugs. Thepresence of the peptides substantially converts the therapeutic druginto an inactive prodrug. The prodrugs do not show significantnon-specific toxicity, but in environments where PSMA is found, theprodrugs become activated upon peptide cleavage, releasing thetherapeutic drug, which then exhibits its inherent non-specifictoxicity.

[0024] PSMA-Specific Peptide

[0025] In one aspect, the invention features prodrugs including apeptide containing an amino acid sequence that includes a cleavage sitespecific for PSMA or an enzyme having a proteolytic activity of PSMA.Prodrugs are designed that can be activated by the pteroylpoly-γ-glutamyl carboxypeptidase (folate hydrolase) activity of PSMA.Gamma glutamyl hydrolase (GGH) is secreted by hepatocytes and by avariety of tumor cell types and GGH activity is present in human serum.Therefore, effective side chain-linked substrates are desirablyspecifically hydrolyzed by PSMA with minimal hydrolysis by GGH.

[0026] The PSMA cleavage site includes at least the dipeptide, X₁X₂.This peptide contains the amino acids Glu or Asp at position X₁. X₂ canbe Glu, Asp, Gln, or Asn. Tripeptides X₁X₂X₃ are also suitable, with X₁and X₂ defined as before, with X₃ as Glu, Asp, Gln or Asn. TetrapeptidesX₁X₂X₃X₄ are also suitable, with X₁₋₃ defined as above, and with X₄ asGlu, Asp, Gln or Asn. Pentapeptides X₁X₂X₃X₄X₅ are also suitable, withX₁₋₄ defined as above, and with X₅ as Glu, Asp, Gln or Asn. HexapeptidesX₁X₂X₃X₄X₅X₆ are also suitable, with X₁₋₅ defined as above, and with X₆as Glu, Asp, Gln or Asn. Further peptides of longer sequence length canbe constructed in similar fashion.

[0027] Generally, the peptides are of the following sequence: X₁ . . .X_(n), where n is 2 to 30, preferably 2 to 20, more preferably 2 to 15,and even more preferably 2 to 6, where X₁ is Glu, Asp, Gln or Asn, butis preferably Glu or Asp, and X₂-X_(n) are independently selected fromGlu, Asp, Gln and Asn. Some preferred peptide sequences are as above,except that X₂-X_(n-1) are independently selected from Glu, and Asp, andX_(n) is independently selected from Glu, Asp, Gln and Asn. The lengthof the peptide can be optimized to allow for efficient PSMA hydrolysis,enhanced solubility of therapeutic drug in aqueous solution, if this isneeded, and limited non-specific cytotoxicity in vitro.

[0028] Among the α-linked dipeptides, Asp-Glu, Asp-Asp, Asp-Asn andAsp-Gln are preferably employed for use in the prodrugs describedherein. Among the all α-linked tripeptides, Glu-Glu-Glu, Glu-Asp-Glu,Asp-Glu-Glu, Glu-Glu-Asp, Glu-Asp-Asp, Asp-Glu-Asp, Asp-Asp-Glu,Asp-Asp-Asp, Glu-Glu-Gln, Glu-Asp-Gln, Asp-Glu-Gln, Glu-Glu-Asn,Glu-Asp-Asn, Asp-Glu-Asn, Asp-Asp-Gln, and Asp-Asp-Asn are preferablyemployed for use in the prodrugs described herein. Tripeptidescontaining Gln or Asn in positions X₂ can also be desirably employed.Longer all α-linked peptides may also be employed for use in theprodrugs described herein, and such peptides with Gln or Asn in anypositions X₂-X_(n) can also be desirably employed.

[0029] Side-Chain Linkages

[0030] PSMA is also able to hydrolyze a variety of side chain-linkedpeptides. Particular side chain-linked, for example, γ-linked peptidesare not specific for PSMA, but can also hydrolyzed by GGH. Somepreferred peptides take advantage of the dual ability of PSMA tohydrolyze certain α- and side-chain linkages between aspartyl, andglutamyl residues.

[0031] Among the side chain-linked dipeptides, Glu*Asp, Glu*Asn,Glu*Glu, Glu*Gln, Asp*Asp, Asp*Glu, Asp*Asn, and Asp*Gln can be employedfor use in the prodrugs described herein. Among the all sidechain-linked tripeptides, Glu*Glu*Glu, Glu*Asp*Glu, Asp*Glu*Glu,Glu*Glu*Asp, Glu*Asp*Asp, Asp*Glu*Asp, Asp*Asp*Glu, Asp*Asp*Asp,Glu*Glu*Gln, Glu*Asp*Gln, Asp*Glu*Gln, Glu*Glu*Asn, Glu*Asp*Asn,Asp*Glu*Asn, Asp*Asp*Gln, and Asp*Asp*Asn can be preferably employed foruse in the prodrugs described herein. Longer peptides which of analogoussequences can also be employed for use in the prodrugs described herein.

[0032] Mixed Peptides

[0033] Some preferred peptides include a PSMA-hydrolyzable, α-linkeddipeptide “cap” that are not substrates for GGH, and are more specificPSMA substrates. Combination α- and side chain-linked PSMA substratescan be highly efficient and specific. For example, Glu*Glu*Glu*Asp-Glu,and Glu*Glu*Glu*Asp-Gln have high stability in serum. Peptidescontaining two α-linkages and two γ-linkages, for example,Asp-Glu*Glu*Asp-Glu can be completely stable to hydrolysis in human andmouse plasma. A number of aspartate- and glutamate-containing linkersare depicted in FIG. 2. These particular linkers can be bonded to aminegroups on therapeutic drugs.

[0034] The peptides listed are among those that are preferred:Glu*Glu*Glu*Asp-Glu, Asp-Glu*Glu*Asp-Glu, and Glu-Glu*Glu*Asp-Glu.Numerous other peptides with mixed α- and side chain linkages andotherwise corresponding to the description herein can be readilyenvisioned and constructed by those of ordinary skill in the art.

[0035] The peptides of the invention are preferably not more than 20amino acids in length, more preferably not more than 6 amino acids inlength. Some peptides which are only two or three amino acids in lengthare quite suitable for use in the prodrugs described herein. Somepreferred amino acid sequences of the invention are linear. However,multiple linkage sites present on dicarboxylic amino acids may also beused to produce branched peptides. These branched peptides could includea therapeutic agent coupled to each amino acid of the peptide chain,such that cleavage of individual amino acids from the peptide chain bythe enzymatic activity of PSMA releases multiple molecules oftherapeutic agent.

[0036] Further examples of the peptides of the invention are constructedas analogs of, derivatives of, and conservative variations on the aminoacids sequences disclosed herein. The term “isolated” as used hereinrefers to a peptide substantially free of proteins, lipids, nucleicacids, for example, with which it is naturally associated. Those ofskill in the art can make similar substitutions to achieve peptides withgreater activity and/or specificity toward PSMA. For example, theinvention includes the peptide sequences described above, as well asanalogs or derivatives thereof, as long as the bioactivity of thepeptide remains. Minor modifications of the primary amino acid sequenceof the peptides of the invention may result in peptides which havesubstantially equivalent activity as compared to the specific peptidesdescribed herein. Such modifications may be deliberate, as bysite-directed mutagenesis or chemical synthesis, or may be spontaneous.All of the peptides produced by these modifications are included herein,as long as the biological activity of the original peptide remains,i.e., susceptibility to cleavage by PSMA.

[0037] Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would also have utility.For example, amino or carboxy terminal amino acids which may not berequired for biological activity of the particular peptide can beremoved. Peptides of the invention include any analog, homolog, mutant,isomer or derivative of the peptides disclosed in the present invention,as long as the bioactivity as described herein remains. All peptideswere synthesized using L-amino acids, and these amino acids arepreferred; however, D-forms of the amino acids can be syntheticallyproduced.

[0038] The peptides of the invention include peptides which areconservative variations of those peptides specifically exemplifiedherein. The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine, alanine,cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine,norleucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, and the like.Neutral hydrophilic amino acids which can be substituted for one anotherinclude asparagine, glutamine, serine, and threonine. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Such conservative substitutions arewithin the definition of the classes of the peptides of the inventionwith respect to X positions which may be any of a number of amino acids.The peptides which are produced by such conservative variation can bescreened for suitability of use in the prodrugs of the inventionaccording to the methods for selecting prodrugs provided herein.

[0039] The peptides of the invention can be synthesized according to anyof the recognized procedures in the art, including such commonly usedmethods as t-boc or fmoc protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C-terminus of the peptide. (see, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well-knownsolid phase peptide synthesis methods described in Merrifield, J. Am.Chem. Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptideSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mM amine/gram polymer.Polyglutamated methotrexate was purchased from Schirks Laboratories.Other analogs were constructed using APA purchased from Sigma Chemical(St. Louis, Mo.). The peptides were synthesized with the appropriateblocking groups on the carboxyl groups, and the APA was coupled to thepeptide using standard coupling chemistry. Such synthetic procedures arewell known to those of ordinary skill in the art.

[0040] On completion of chemical synthesis, the peptides can bedeprotected and cleaved from the polymer by treatment with liquid HF-10%anisole for about ¼ to 1 hour at o° C. After evaporation of thereagents, the peptides are extracted from the polymer with 1% aceticacid solution which is then lyophilized to yield the crude material.This can normally be purified by such techniques as gel filtration onSephadex G-15 using 5% acetic acid as solvent. Lyophilization ofappropriate fractions of the column will yield the homogeneous peptideof peptide derivatives, which can then be characterized by such standardtechniques as amino acid analysis, thin layer chromatography, highperformance liquid chromatography, ultraviolet absorption spectroscopy,molar rotation, solubility, and quantitated by solid phase Edmandegradation.

[0041] The invention encompasses isolated nucleic acid moleculesencoding the PSMA-specific peptides of the invention, vectors containingthese nucleic acid molecules, cells harboring recombinant DNA encodingthe PSMA-specific peptides of the invention, and fusion proteins whichinclude the PSMA-specific peptides of the invention. Especiallypreferred are nucleic acid molecules encoding the polypeptides describedherein.

[0042] The PSMA-specific peptides are cleaved by PSMA to yield at least5 picomoles, preferably at least 10 picomoles, and most preferably atleast 15 picomoles of cleaved peptide per minute per milligram of PSMA.Desirably, the peptides are highly selective towards cleavage by PSMA,so that cleavage by other purified extracellular proteases is minimized.The peptides disclosed herein are cleaved by extracellular proteasesother than PSMA to yield not more than 4.0 picomoles, preferably notmore than 2.0 picomoles, and more preferably not more than 1.0 picomoleof cleaved peptide per minute per milligram of purified extracellularnon-PSMA proteases. The peptides described herein are also stable towardcleavage in sera. The peptides containing this sequence yield at most5%, preferably at most 2.5% and more preferably at most 1.0% cleavedpeptide from uncleaved peptide in human serum over a 24-hour period.

[0043] Prodrug Compositions

[0044] A wide variety of entities can be linked to the a-amino terminus,the α-carboxy terminus, or the side chain of the peptide, preferably atX₁, but also at any position from X₁ to X_(n−1). In some preferredembodiments, linkage between the entities and the peptide takes place atX₁, at either the amino terminus, or at the side chain.

[0045] Notably, therapeutic drugs can be linked to these positions,creating prodrugs. The therapeutic drugs that may be used in theprodrugs of the invention include any drugs which can be directly orindirectly linked to the PSMA-specifically cleavable peptides of theinvention. Hydrolytic processing of prodrugs by PSMA results in a finalproduct consisting of a therapeutic drug coupled to an amino acid suchas aspartate or glutamate. Preferred therapeutic drugs incorporateaspartic, glutamic acid or some other dicarboxylic acid into theirstructure and still maintain their therapeutic effect, for example,cytotoxicity. In this way, advantage is taken of the PSMA-specificity ofthe cleavage site, as well as other functional characteristics of thepeptides of the invention. Preferred drugs are those that contain anacidic amino acid, for example Asp or Glu. The presence of an amino acidin the drug allows the formation of an amide bond between the drug andthe peptide. This bond serves as the cleavage site for PSMA. As notedabove, the peptides of the invention can be used to activate therapeuticdrugs at PSMA producing tissue. The peptides which are useful in theprodrugs of the invention are those described above.

[0046] Certain therapeutic drugs contain acidic amino acids. Examples ofthese include methotrexate, ralitrexed (Tomudex), edatrexate, and 5,10dideaztetrahydrofolate (Lometrexol). Other therapeutic drugs arerequired to have acidic amino acids introduced by chemical orbiochemical synthesis, for example, sesquiterpene-γ-lactones such asthose belonging to the guaianolide, inuchineolide, germacranolide, andeudesmanolide families of sesquiterpenoids. These include estafiatin,grossheimin, inuchinenolide, arglabin, thapsigargin and theirderivatives, such as thapsigargicin and many others known to thoseskilled in the art. Thapsigargin and its derivatives are believed to actby inhibiting the SERCA pump found in many cells. Other classes ofagents include acidic amino acid containing derivatives of theantifolate trimetrexate, and the anthracyclines antibiotics containingan amino sugar such as doxorubicin, daunorubicin, epirubicin(4-epidoxorubicin), idarubicin (4-demethoxydaunomycin) and the like.These drugs intercalate into polynucleotides and interfere withreplication processes. An additional class of agents would includederivatives of the taxane class of agents (examples of this class aretaxol and taxotere). Amino acid-containing derivatives of these agentsmaintain therapeutic efficacy.

[0047] Preferably, therapeutic drugs are linked to the peptide eitherdirectly or indirectly, through a linker group. The direct linkage canbe made conveniently through an amide bond, for example. If therapeuticdrugs are linked to the peptide through the α-amino group of X₁, anamide bond is conveniently created with a carboxyl present on thetherapeutic drug, or with a carboxyl present on any linker. Iftherapeutic drugs are linked to the peptide through the side chain- orα-carboxyl of X₁ or any other amino acid in the peptide, an amide bondis conveniently created with an amino group present on the therapeuticdrug, or with an amino group present on any linker.

[0048] The linker may be connected to the therapeutic drug through anyof the bond types and chemical groups known to those skilled in the alt.Therapeutic drugs can also be coupled directly to the α-amine of anamino acid of peptides via a linker.

[0049] The linker can either remain attached to the drug or be cleavedoff. In embodiments in which the linker remains attached to the drug,the linker can be any group which does not substantially inhibit thenon-specific toxicity of the drug after cleavage from the peptide.Suitable linkers are primary amine containing alkanoyl, alkenoyl, andarenoyl substituents. Examples of such linkers areCO—(CH═CH)_(n1)—(CH₂)_(n2)—Ar—NH₂, CO—(CH₂)_(n2)—(CH═CH)_(n1)—Ar—NH₂,CO—(CH₂)_(n2)—(CH═CH)_(n1)—CO—NH—Ar—NH₂ andCO—(CH═CH)_(n1)—(CH₂)_(n2)—CO—NH—Ar—NH₂ and substituted variationsthereof, where n1 and n2 are from 0 to 5, and Ar is any substituted orunsubstituted aryl group. Substituents which may be present on Arinclude short and medium chain is alkyl, alkanoxy, aryl, aryloxy, andalkenoxy groups, nitro, halo, and primary secondary or tertiary aminogroups, as well as such groups connected to Ar by ester or amidelinkages. Amino acids can also serve as linkers. A dicarboxylic acidlinker can be used, such as the 12-carbon linker 12-carboxydodecanoate,shown, for example, for (12-CDT-Asp) in FIG. 2. This analog can then belinked via either the α-carboxyl or side-chain carboxyl to a longerpeptide chain.

[0050] In other embodiments, the linker is self-cleaving. Self-cleavinglinkers are those which are disposed to cleave from the drug after thecleavage of the peptide by PSMA. The linkers generally contain primaryamines which form amide bonds to the carboxy terminus of the peptidesequence. The linkers can also contain a carboxylic acid which forms anamide bond to a primary amine found on the drug.

[0051] In such embodiments, the linker is not required to benon-interfering with the non-specific toxicity of the drug, as long asit is cleaved within a period of time short enough to allow the drug toremain localized where it has been activated, or within a period of timeshort enough to prevent inactivation by any means.

[0052] The linker may remain on the therapeutic drug indefinitely aftercleavage, or may be removed soon thereafter, either by further reactionswith external agents, or in a self-cleaving step. Self-cleaving linkersare those linkers which can intramolecularly cyclize and release thedrug, or undergo spontaneous S_(N)I solvolysis and release the drug uponpeptide cleavage. Such linkers are for example 2, 2-dialkyl-2-(2-anisyl)acetic acid, described in Atwell et al., J. Med. Chem., 37:371-380,(1994), and p-amidobenzyloxycarbonyl, described in Carl et al., J. Med.Chem., 24:479-480, (1981). Further useful examples are provided in thesereferences. Other materials such as detectable labels or imagingcompounds can be linked to the peptide. Groups can also be linked to thecarboxy side chains of X₁ to X_(n−1), including such moieties asantibodies, and peptide toxins, including the 26 amino acid toxin,melittin and the 35 amino acid toxin, cecropin B, for example. Both ofthese peptide toxins have shown toxicity against cancer cell lines.

[0053]FIG. 1 is a structure of a particular embodiment of aPSMA-activated TG prodrug. DBTG refers to 8-O-debutanoylthapsigargin,which is linked via the oxygen atom to the remainder of the prodrug asshown. The portion of the molecule labeled as E*12-ADT is the 12-aminododecanoate side chain. Thus, preferred substrates combine thespecificity of the α-linkage with the enhanced efficiency of theγ-linkage. The longer-length, negatively-charged, substrates can servetwo additional purposes: first, they help to make highly lipophilictoxins, for example, TG analogs, more water soluble; second, the highlycharged prodrug will be less likely to cross the plasma membrane,further limiting non-specific cytotoxicity.

[0054] The following prodrugs are particularly preferred:

[0055] (1)12ADT-Glu* Glu* Glu*Asp-Glu (4)12ADT-Asp-Glu* Glu*Asp-Glu(2)12ADT*Glu-Glu*Glu*Asp-Glu (5)12CDT-Asp-Glu*Glu*Asp-Glu (3)12CDT-Glu*Glu*Glu*Asp-Glu

[0056] The prodrugs are hydrolyzed by PSMA and release the correspondingAsp- or Glu-containing TG analogs or the TG analog itself, and also lackpotent cytotoxicity when not metabolized by PSMA. Non-PSMA producingTSU-Pr1 human prostate cancer cell line is exposed to each of theprodrugs at doses that are approximately 50-times the LD₅₀ for thecorresponding free TG analog. Against the TSU prostate cancer cell line,12ADT-Glu has an LD₅₀ value for killing of ˜50 nM.

[0057] The prodrugs are hydrolyzed by PSMA and have a dose-responsiveability to kill PSMA-producing LNCAP and CWR22R cells in vitro, basedupon loss of clonogenic abilities. The activity of these cell lines isapproximately 13 pmoles NAAG hydrolyzed/min/mg protein for LNCaP andapproximately 20 pmoles NAAG hydrolyzed/min/mg protein for CWR22R cells,using radiolabeled ³H-NAAG. These prodrugs are tested against TSU cellsthat have been transduced with a lentiviral vector carrying the PSMAgene. This TSU-PSMA cell line produces amounts of PSMA that are similarto LNCaP as determined by Western Blot. The activity of the PSMA fromthis line is comparable to the LNCaP and CWR22R lines (that is,approximately 18 pmoles NAAG hydrolyzed/min/mg protein). This TSU-PSMAline is used to determine the therapeutic index by comparing cytotoxicactivity of the prodrugs against this PSMA-producing line and the wildtype TSU cells. Using these data, LD₅₀ values for all the testedcompounds is calculated. To be considered selective, the preferredprodrugs have a >20-fold difference in ability to kill TSU-PSMA vs. TSUwild type cells.

[0058] The prodrugs of the invention are not taken up by the cells, butare cleaved extracelullarly by PSMA to yield at least 5 picomoles,preferably at least 10 picomoles, and more preferably at least 15picomoles of therapeutic drug per minute per milligram of PSMA.Preferably, the prodrugs of the invention are cleaved by extracellularproteases other than PSMA to yield not more than 4.0 picomoles,preferably not more than 2.0 picomoles, and more preferably not morethan 1.0 picomole of therapeutic drug per minute per milligram ofpurified extracellular non-PSMA proteases. The prodrugs of the inventionyield at most 5%, preferably at most 2.5%, and more preferably at most1.0% of prodrug as therapeutic drug in human serum over a 24-hourperiod.

[0059] The prodrugs of the invention may also comprise groups whichprovide solubility to the prodrug as a whole in the solvent in which theprodrug is to be used. Most often the solvent is water. This feature ofthe invention is important in the event that neither the peptide nor thetherapeutic drug is soluble enough to provide overall solubility to theprodrug. These groups include polysaccharides or other polyhydroxylatedmoieties. For example, dextran, cyclodextrin, starch and derivatives ofsuch groups may be included in the prodrug of the invention.

[0060] Methods of Treatment Using Prodrugs

[0061] The invention also provides methods of treating PSMA-producingcell proliferative disorders of the invention with the prodrugs of theinvention. Hydrolytic processing of prodrugs by PSMA results in a finalproduct consisting of a therapeutic drug or a therapeutic drug coupledto an amino acid such as aspartate or glutamate. Preferred therapeuticdrugs incorporate aspartic, glutamic acid or some other dicarboxylicacid into their structure and still maintain their therapeutic effect.Prodrugs can be tested for cytotoxicity against PSMA-producing LNCaP,CWR22R and the TSU-PSMA and wild type TSU human cancer cells.

[0062] The prodrugs of the invention and/or analogs or derivativesthereof can be administered to any host, including a human or non-humananimal, in an amount effective to treat a disorder.

[0063] The prodrugs of the invention can be administered parenterally byinjection or by gradual infusion over time. The prodrugs can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. Preferred methods fordelivery of the prodrug include intravenous or subcutaneousadministration. Other methods of administration will be known to thoseskilled in the art.

[0064] Preparations for parenteral administration of a prodrug of theinvention include sterile aqueous or non-aqueous solutions, suspensions,and emulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives can also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases, and the like.

[0065] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. ThePSMA-specific prodrugs of the invention are useful in treatingmalignancies of the various organ systems. Essentially, any disorderwhich is etiologically linked to PSMA expression could be consideredsusceptible to treatment with a PSMA-specific prodrug. One such disorderis a malignant cell proliferative disorder, for example. The term“therapeutically effective amount” as used herein for treatment of cellproliferative disorders refers to the amount of prodrug sufficient tocause a reduction in the number of unwanted cells. The term“therapeutically effective” therefore includes the amount of prodrugsufficient to prevent, and preferably reduce by at least 25%, and morepreferably to reduce by 90%, the number of unwanted cells. The dosageranges for the administration of prodrug are those large enough toproduce the desired effect. Generally the dosage will vary with age,condition, sex, and extent of the disorder in the subject, and can bedetermined by one skilled in the art. The dosage can be adjusted by theindividual physician in the event of any contraindications. In anyevent, the effectiveness of treatment can be determined by monitoringtumor ablation.

[0066] Method of Producing Prodrugs

[0067] The invention, in another aspect, provides a method of producingthe prodrugs of the invention. Tis method involves linking atherapeutically active drug to a peptide of the invention. Such peptidesare described above. After the drug and peptide are linked to produce atherapeutic prodrug composition, the non-specific toxicity of the drugis substantially inhibited. In certain embodiments, the peptide islinked directly to the drug. In other embodiments, the peptide isindirectly linked to the drug, the linkage occurring through a linker.In each case the amino terminus of the peptide is used for linking. Thedrug can be linked to the α-amine of the amino terminal amino acid or itcan be linked to a carboxyl side-chain of an acidic amino acid at theamino terminus of the peptide, or at any position from X₂ to X_(n−1)except when n is 2. That is, in an amino acid sequence X₁X₂ . . .X_(n−1), the link is established through X₁ or X₂ to X_(n−1), preferablythrough X₁. The therapeutic drug can contains a primary amine group or acarboxyl group to facilitate the formation of an amide bond with thepeptide. Many acceptable methods of coupling carboxyl and amino groupsto form amide bonds are known to those of skill in the art.

[0068] The bonds of the amino acids in the peptide are sequentiallycleaved by PSMA, releasing the therapeutic drug. Suitable linkersinclude any chemical group which contains a primary amine or carboxylgroup. The linkers for use in the present invention include amino acids,primary amine- or carboxyl-containing alkyl, alkenyl or arenyl groups.

[0069] The connection between the linker and the therapeutic drug may beof any type known in the art, preferably covalent bonding. The linkergroup may remain attached to the therapeutic drug if its attachment doesnot significantly reduce the non-specific toxicity of the drug. Incertain embodiments, the linker is a cleavable linker, which may becleaved either by an external agent, or it may be a self-cleavinglinker. External agents which may effect cleavage of the linker includeenzymes, proteins, organic or inorganic reagents, protons and any otheragents which do not affect the non-specific toxicity of the drug orprodrug.

[0070] In certain embodiments, the linker comprises an amino acidsequence. The sequence may be of any length, but is preferably between 1and 10 amino acids, most preferably between 1 and 5 amino acids inlength. Preferred amino acids are glutamate, aspartate, glutamine,asparagine, or amino acid sequences containing these amino acids,especially at their amino termini, although conservative variations ofthese amino acids may also be utilized. More preferably, the linkerincludes glutamate or aspartate.

[0071] Other groups may be added to the prodrugs of the invention,including those which render the prodrug soluble in water. These groupsinclude polysaccharides or other polyhydroxylated moieties. For example,dextran, cyclodextrin and starch may be included in the prodrug of theinvention.

[0072] Method of Screening Tissue

[0073] In another aspect the invention provides a method of detectingPSMA-producing tissue using the peptides of the invention, as describedabove. The method is carried out by contacting a detectably labeledpeptide of the invention with target tissue for a period of timesufficient to allow PSMA to cleave the peptide and release thedetectable label. The detectable label is then detected. The level ofdetection is then compared to that of a control sample not contactedwith the target tissue. Many varieties of detectable label areavailable, including optically based labels, such as chromophoric,chemiluminescent, fluorescent or phosphorescent labels, and radioactivelabels, such as alpha, beta or gamma emitting labels. Examples offluorescent labels include amine-containing coumarins such as7-amino-4-methylcoumarin, 7-amino-4-trifluoromethyl, and otheramine-containing fluorophores such as 6-aminoquinoline, and rhodamines,including rhodamine 110. Other examples of fluorescent labels includethose containing carboxyl moieties such as FITC. Examples of radioactivelabels include beta emitters such as ³H, ¹⁴C and ¹²⁵I. Examples ofchromophoric labels (those that have characteristic absorption spectra)include nitroaromatic compounds such as p-nitroaniline. Examples ofchemiluminescent labels include luciferins such as6-amino-6-deoxyluciferin.

[0074] Preferably, the choice of detectable label allows for rapiddetection and easily interpretable determinations. Detectable labels foruse in the invention preferably show clearly detectable differencesbetween detection from the cleaved and uncleaved state.

[0075] The invention provides a method for detecting a cellproliferative disorder which comprises contacting a PSMA-specificpeptide with a cell suspected of having a PSMA-production associateddisorder and detecting cleavage of the peptide. The peptide reactivewith PSMA is labeled with a compound which allows detection of cleavageby PSMA. For purposes of the invention, a peptide specific for PSMA maybe used to detect the level of enzymatically active PSMA in cellmembranes, and potentially in saliva, blood, or urine. Any specimencontaining a detectable amount of antigen can be used. The level of PSMAin the suspect cell can be compared with the level in a normal cell todetermine whether the subject has a PSMA-production associated cellproliferative disorder. Preferably the subject is human.

[0076] Method of Screening Prodrugs

[0077] The invention also provides a method of selecting potentialprodrugs for use in the invention. The method generally consists ofcontacting prodrugs of the invention with PSMA-producing tissue andnon-PSMA producing tissue in a parallel experiment. “PSMA-producingtissue” as used herein is tissue that produces at least 1 ngenzymatically active PSMA per gram of tissue, or at least 1 ng ofenzymatically active PSMA/10⁶ cells/24 hours from cells. The prodrugswhich exert toxic effects in the presence of PSMA-producing tissue, butnot in the presence of non-PSMA producing tissue are suitable for theuses of the invention. In other words, the LC₅₀ concentration of theprodrug in the presence of PSMA-producing tissue is at least 3 timeslower, more preferably at least 20 times lower, and most preferably atleast 100 times lower than the LC₅₀ concentration of the prodrug in thepresence of non-PSMA producing tissue.

[0078] Method of Determining PSMA Activity

[0079] The invention also provides a method of determining the activityof PSMA. The method generally consists of contacting detectably labeledprodrugs of the invention with samples may come from fluid drawn fromPSA-producing tissue, from tissue culture media, from serum, saliva orurine, or any source which contains PSMA. The cleavage of peptide whichtakes place by PSMA results in the release of a detectable label, whichis subsequently detected. This detection level is compared to thedetection level which is found upon performing a parallel experiment inwhich the PSMA-containing sample is a standard solution made up frompurified PSMA as described, for example, in Lapidus et al, Prostate,(2000) 45:350-354. This comparison results in a determination of theactivity of the PSMA which is present in the sample, given a correctionfor any differences in PSMA concentration which may exist. Suchcorrection may be accomplished directly by adjusting the concentrationsof the standard and sample solutions to match each other or bymathematical correction means.

[0080] Method of Imaging Tissue

[0081] The invention in another aspect, provides a method of imagingsoft tissue or bone metastases by providing peptides of the inventionlinked to lipophilic imaging labels that can be detected by imagingtechniques, for example, positron emission tomography (PET). This methodis accomplished generally by administering a peptide of the inventionlinked to a primary amine-containing lipophilic label to a subjecthaving or suspected of having a PSMA-producing associated cellproliferative disorder. The peptide is selectively cleaved from thelipophilic imaging label where enzymatically active PSMA occurs in thesubject (i.e., PSMA producing tissues). The lipophilic imaging label isthen drawn into the membranes of cells in the vicinity. After a periodof time sufficient to allow cleavage of the peptide by PSMA, and toallow the uncleaved peptide to be sufficiently cleared from the subjectto allow reliable imaging, the subject is imaged. The lipophilic labelaccumulates in the soft tissue or bone that produces PSMA, and allows adiagnosis of the subject. Suitable labels for PET scanning areradionuclides such as ¹⁸F, ¹¹C, ¹³N and ¹⁵O, and any other positronemitters known in the art. Lipophilicity can be engineered into thelabel by introducing the label into lipophilic fragments or moietiesknown to those in the art, by methods known to those skilled in the art.

[0082] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES

[0083] The following examples illustrate the preparation and propertiesof certain embodiments of the invention.

Example 1 Determination of PSMA Hydrolysis of Peptides

[0084] The extent to which various peptide substrates are hydrolyzed byPSMA was determined by synthesizing a series of methotrexate analogsincluding the4-N[N-2,4-diamino-6-pteridinyl-methyl)-N-methylamino-benzoate] (APA)portion of methotrexate coupled to a variety of peptides.Poly-glutamated methotrexate is available from Schirks Laboratories inJona, Switzerland. The coupling of APA to the N-terminal amine ofγ-linked polyglutamates does not inhibit sequential PSMA hydrolysis.

[0085] A series of α-linked peptides were investigated for PSMAhydrolysis. The results are shown in Table 1. The table shows thepercentage of a-linked methotrexate analogs hydrolysed to methotrexateafter being incubated with PSMA for 24 hours and for 48 hours. In Tables1-3, to simplify identification of the type of amino acid linkagesemployed, α-linkages are denoted by a hyphen (for example, Glu-Glu) andall side-chain linkages are denoted by a star (for example, Glu*Glu).TABLE 1 Methotrexate Analogs Hydrolyzed by PSMA (α-linked) % hydrolysisafter 24 % hydrolysis after 48 Methotrexate Analog hours hoursAPA-Glu-Glu 0.0 0.0 APA-Glu-Glu-Glu 0.0 0.0 APA-Asp 0.0 0.0 APA-Glu-Asp0.0 0.0 APA-Asp-Glu 21.5 45.6 APA-Glu-Gln 0.0 0.0 APA-Asn-Glu 0.0 0.0poly*Glu 96.1 100

[0086] Substrate specificity for the pteroyl poly-y-glutamylcarboxypeptidase (folate hydrolase) activity of PSMA was determined bysynthesizing a series of γ-linked methotrexate analogs and assaying themfor hydrolysis by PSMA. These results are shown in Table 2. TABLE 2Methotrexate Analogs Hydrolyzed by PSMA (γ-linked) % hydrolysis after 24% hydrolysis after 48 Methotrexate Analog hours hours APA-Glu*Asp 38.384.6 APA-Glu*Asp*Glu 37.8 85.5 APA-Glu*dGlu 0.0 0.0 APA-Glu*Gln 48.2APA-Glu*GABA 0.0 0.0 APA- 40.9 77.7 Glu*Glu*Glu*Glu*Asp poly*Glu 96.1100

[0087] In Table 2, GABA is gamma aminobutyric acid. The poly-Glu is ally-linked in each of Tables 1 and 2.

[0088] In order that the prodrugs can be administered systemically viathe blood, their stability in sera must be sufficiently high to avoidhydrolysis or other degradative processes which may occur prior to theprodrugs reaching their target tissues. In addition to GGH activity, ahuman blood plasma glutamate carboxypeptidase (PGCP) that hassignificant sequence homology to PSMA and glutamate carboxypeptidaseactivity has been identified. Therefore, to assay hydrolysis by GGH andother serum carboxypeptidases like PGCP, the APA-Glu*Glu*Glu*Glu*Asp,APA-Glu*Glu*Glu*Glu*Gln, and APA-Glu*Glu*Glu*Glu*Glu substrates wereincubated in human and mouse plasma and hydrolysis determined by HPLCanalysis after an 18-hour incubation, as shown in Table 3. As noted, GGHefficiently hydrolyzes poly-γ-glutamate chains as well aspoly-γ-glutamated folate and methotrexate. GGH, however, is unable tohydrolyze α-linked peptides. An α-linked analog that was found to behydrolyzable by PSMA, APA-Asp-Glu, was tested for stability in humanserum, as shown in Table 3. TABLE 3 Hydrolysis of PSMA Substrates byPurified PSMA and Stability in Plasma 18 hour Incuba- tion in Mouse 18hour Incubation in Plasma Human Plasma % % PSMA % APA- APA- hydrolysisGlu or - % Prodrug Glu Substrate 24 h 48 h Asp Remaining or -Aspα-linked APA-Asp-Glu 70 99 1 99 ND APA-Glu-Glu 0 20 ND ND ND γ-linkedAPA- 98 100 9 20 72 Glu*Glu*Glu*Glu*Asp APA- 97 100 6 26 62Glu*Glu*Glu*Glu*Gln APA- 96 100 11 11 68 Glu*Glu*Glu*Glu*Glu α-,γ-linked APA-Glu*Glu*Glu*Asp- 30 65 23 57 5 Glu APA-Glu*Glu*Glu*Asp- 510 13 76 ND Gln APA-Asp-Glu*Glu*Asp- 36 77 0 100 2 Glu

[0089] In Tables 1-3, to simplify identification of the type of aminoacid linkages employed, α-linkages are denoted by a hyphen (for example,Glu-Glu) and all side-chain linkages are denoted by a star (for example,Glu*Glu). The column labeled “% PSMA Hydrolysis” refers to thepercentage of hydrolysis of a substrate to APA-Asp or to APS-Glu bypurified PSMA. The column labeled “% APA-Asp or -Glu” refers to thepercentage of hydrolysis of substrate to APA-Asp or APA-Glu. The columnlabeled “% Prodrug Remaining” refers to the area of the HPLC peakattributable to starting material divided by the areas of HPLC peaksattributable to starting material, intermediate products, and finalproducts. The hydrolysis of substrates in mouse plasma did not displayany peaks attributable to intermediate products, but only showedstarting material and APA-. “ND” means “not determined,” in that theexperiment was not performed.

[0090] As shown in Table 3, the side chain-linked dipeptides APA-Glu*Aspand APA-Glu*Gln were significantly hydrolyzed by PSMA. The peptidesAPA-Glu*Glu*Glu*Glu*Asp and APA-Glu*Glu*Glu*Glu*Gln, containing aγ-linked Asp or Gln were both hydrolyzed by PSMA to yield APA-Glu (thatis, methotrexate) to a greater extent after 24 hours than any of theγ-linked dipeptide analogs. These analogs, however were less efficientsubstrates when compared to APA-Glu*Glu*Glu*Glu*Glu, the polyglutamatedmethotrexate analog with similar γ-glutamyl chain length, as shown inTable 3.

[0091] For APA-Glu*Glu*Glu*Glu*Asp, APA-Glu*Glu*Glu*Glu*Gln, andAPA-Glu*Glu*Glu*Glu*Glu, >75% of the starting material was hydrolyzed tomethotrexate or to other intermediate length species, consistent withsequential hydrolysis by the exopeptidase activity of GGH present inhuman serum, as shown in Table 3. In mouse plasma, approximately 60-75%conversion of each analog directly to APA-Glu (that is, methotrexate)was observed after 18 hours, as shown in Table 3. The addition ofp-hydroxymercuribenzoate, a non-specific inhibitor of GGH and othercysteine proteases, resulted in complete inhibition of hydrolysis.

[0092] After an 18 hour incubation in human serum, HPLC analysisdemonstrated no significant hydrolysis of the dipeptide APA-Asp-Glu,suggesting the inability of serum GGH and other serum carboxypeptidaseslike PGCP to cleave alpha-linked acidic peptides.

Example 2 Preparation of Thapsigargin Analogs

[0093] The starting material for all the synthesized analogs is8-O-debutanoylthapsigargin, which is easily available by triethylaminecatalyzed methanolysis of thapsigargin. Removal of the butanoyl resultsin loss of cytotoxic activity with an LD₅₀ of >50 μM compared to <100 nMfor thapsigargin. Anhydrides of dicarboxylic acids of various lengthsafforded analogs in which the acyl group attached to the O-8 ended in afree carboxylic acid. A dicyclohexylcarbodiimide (DCCI) promotedcoupling of a 2,4-diaminoarene to the carboxylic acid analogs affordsthe derivatives in which contain a primary aromatic amine as a potentialcoupling point for additional moeities.

[0094] Another type of thapsigargin derivative has been prepared byreacting 8-O-debutanoylthapsigargin with a 4-aminophenyl aliphaticcarboxylic acid like 4-aminocinnamic acid, 3-(4-aminophenyl) propionicacid, or 4-(4-aminophenyl)butanoic acid in the presence of DCCI and4-dimethylaminopyridine. The aromatic amino group had previously beencoupled to a boc-protected -amino acid like glutamine or leucine bystandard techniques. After deprotection of the amino group by standardtechniques the thapsigargin derivative can be coupled to the peptide.

[0095] The synthesis of thapsigargin analogs was performed generally asfollows. Unless otherwise stated all reactions were performed at roomtemperature, and the mixtures filtered and concentrated in vacuo withcolumn chromatography performed over silica gel 60, (0.040-0.063,Merck). Each structure was further proven by ¹³C and ¹H NMR spectroscopyand mass spectrometry. The NMR spectra have been recorded on an AF200XBruker spectrometer in deuterated solutions using tetramethylsilane asan internal standard. In all the spectra, the signal originating in theacetyl, angeloyl, butanoyl, and octanoyl residues have been found aspreviously reported (Christensen, et al. Phytochemistry. 23:1659-63,(1984)), and are not reported. The ¹H NMR spectra were recorded at 200MHz. The signals of H-9′ have, in many cases, been overlapped by signalsfrom the protons in the octanoyl residue. The ¹³C NMR spectra wererecorded at 50 MHz. In the ¹³C NMR spectra the assignments of signalswith similar chemical shift values might be interchanged. The signalsoriginating in C-2 and C-6 are hidden by the signals of chloroform, buthave been visualized in a few cases by recording the DEPT spectra. Thesmall amounts of compounds available have in some cases precluded theobservation of signals of poor intensities.

[0096]FIG. 3 shows a general synthetic scheme for the production of thetitle compounds of this example. On of the ordinary skill in the art oforganic synthesis, particularly peptide synthesis, will recognize theabbreviations given for various reagents, and will also readily be ableto derive appropriate reaction conditions, in light of not only theknowledge and abilities of one of ordinary skill in the art, but also ofthe more detailed procedures given herein.

[0097] A glutamate-containing TG analog, 12ADT-Glu, has been synthesizedaccording to the synthetic method outlined in FIG. 3. According to thereactions illustrated in the synthetic method of FIG. 3, DIPEA isdiisopropylethylamine, DCCI is dicyclohexylcarbodiimide, DMAP is4-dimethylaminopyridine, and TEA is trifluoroacetic acid.

[0098] The TG analog was synthesized by coupling the primary amine of12ADT to the y-carboxyl of glutamate, leaving the a-carboxyl ofglutamate free to link to other amino acids, as shown in FIG. 2. The12-ADT*Glu is a potent, cell-proliferation independent inducer of theapoptotic death of prostate cancer cells (LD₅₀ is approximately 50 nM).The corresponding analog consisting of 12-ADT linked to the a-carboxylof glutamate (that is, 12ADT-Glu) is also a suitable analog, as shown inFIG. 2. The prodrug containing 12ADT*Glu coupled to the longer aminoacid chain (that is, 12ADT*Glu-Glu*Glu*Asp-Glu) can also be a suitablesubstrate. The 12ADT-Glu* Glu* Glu*Asp-Glu prodrug may also be cleavedby PSMA, which may cleave the last peptide bond in the absence of a freeα-carboxyl. TG can be coupled directly to the amine of glutamate via a12-carbon dicarboxylic acid linker (12CDT-Glu), as shown in FIG. 2, toprovide a further useful substrate. This analog can then he linked viaeither the α- or γ-carboxyl to a peptide chain.

Example 3 Preparations of N-L-leucyl-6-aminohexanoyl-,N-L-leucyl-12-amino dodecanoyl-, N-D-leucyl-12-amino dodecanoyl-, andN-L-alanyl-12-amino dodecanoyl-8-O-debutanoylthapsigargins (L-6-AHT.L-12-ADT, LD-12-ADT and A-12-ADT)

[0099]FIG. 5 shows a general synthetic scheme for the production of thetitle compounds of this example. On of the ordinary skill in the art oforganic synthesis, particularly peptide synthesis, will recognize theabbreviations given for various reagents, and will also readily be ableto derive appropriate reaction conditions, in light of not only theknowledge and abilities of one of ordinary skill in the art, but also ofthe more detailed procedures given herein.

Example 4 Preparation ofN-L-y-glutamyl-12-aminododecanoyl-8-O-debutanoyl Thapsigargin(EG-12-ADT)

[0100]FIG. 6 shows a general synthetic scheme for the production of thetitle compound of this example. On of the ordinary skill in the art oforganic synthesis, particularly peptide synthesis, will recognize theabbreviations given for various reagents, and will also readily be ableto derive appropriate reaction conditions, in light of not only theknowledge and abilities of one of ordinary skill in the art, but also ofthe more detailed procedures given herein.

Example 5 In Vivo Administration of Prodrugs

[0101] The in vivo administration of the prodrugs described herein isinitially carried out in athymic nude mice (n=3/group), which willreceive increasing doses of prodrug subcutaneously daily for 5 days todetermine tolerable doses for tumor efficacy studies. Additional animalsare given similar doses intravenously to further determine toxicity.

[0102] Athymic nude mice are inoculated subcutaneously with TSU-PSMAcells. In one set of experiments, tumor-bearing animals (n=10/group) aregiven prodrug subcutaneously with tumor volume measured twice weekly andcompared to vehicle treated tumor-bearing controls. Animals are treatedwith prodrugs daily×5 for a period of 4 weeks or until toxicity or untiltumors are >2 cc. At the end of each experiment, animals are euthanizedand tumors recovered and weighed. Toxicity is assessed twice weekly byvisual inspection and body weights. Animals that lose >15% body weightare sacrificed. To assess specificity of PSMA-mediated prodrughydrolysis, a second set of experiments on animals is performed, inwhich animals are inoculated in one flank with TSU-PSMA cells andsimultaneously inoculated in the opposite flank with wild type TSUcells. These animals are then treated with prodrug to determine ifnon-specific activation by the wild type tumor cells is occurring.Prodrugs that are active against TSU-PSMA are then tested for efficacyagainst other PSMA-producing human prostate cancer xenografts (that is,LNCaP and CW22R).

[0103] To assay the products of PSMA and/or non-specific hydrolysis ofthe TG prodrugs in vivo, prodrugs are labeled using [³H], as describedin Christensen et al., Bioorg. Medicinal Chemistry, 7:1273-80, (1999).HPLC separation of proteolytic products is performed by injectingsamples onto a C-18 reverse phase column and gradient eluted withincreasing concentrations of acetonitrile/0.1% TFA. The tritiated [³H]TG analog coupled products are detected by an inline radioactive flowdetector and by monitoring absorbance at 215 μm. In this way mice areinjected with radiolabeled prodrug, and at various time points animalsare sacrificed. Extracts from serum, PSMA-positive tumors, and organssuch as liver, kidney, and spleen are made. These extracts are analyzedby HPLC with inline radioactive flow to determine presence of the freeTG analog and TG analog coupled to peptide. These results are used toguide additional dosing studies.

Example 6 Selectivity of a PSMA-Targeted Prodrug

[0104] The methotrexate analog sequence APA-Glu*Glu* Glu*Glu*Asp wascontacted with the non-PSMA producing TSU cancer cell line in variousamounts. Exogenous PSMA (10 micrograms/mL) was added to the media andthe percent inhibition of clonogenic survival was monitored. The resultsare shown in FIG. 4. FIG. 4 shows that there is selectivity of thismethotrexate analog sequence.

Example 7 Toxicity of Methotrexate Analogs

[0105] A number of methotrexate analogs were studied for their effect onclonogenic survival of TSU cancer cells. One such analog is L-asparticacid,N-[4-[[(2,4-diamino-5-ethyl-6-quiazolinyl)methyl]amino]benzoyl]-sesquihydrate,also known as NSC 184692, available from the NCI DevelopmentalTherapeutics Branch. Another compound is L-aspartic acid,N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quiazolinyl)methyl]amino]benzoyl]-dihydrate,also known as NSC 132483. Another compound is L-aspartic acid,N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]-monohydrate,also known as NSC 134033.

[0106] The loss of clonogenic survival following treatment of TSU cancercells with NSC 184692 and 132483 has been investigated after 48 hours ofexposure, and the results are listed in Table 4. TABLE 4 Loss ofClonogenic Survival After Exposure to Methotrexate Analogs Concentration(μmolar) NSC 184692 NSC 132483 5 93% 78% 1 88% 50% 0.5 82% 39% 0.1 57%14% 0.05 44%  0% 0.01 17%  0%

Other Embodiments

[0107] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A composition comprising a prodrug, the prodrugcomprising a therapeutically active drug; and a peptide comprising anamino acid sequence having a cleavage site specific for an enzyme havinga proteolytic activity of prostate specific membrane antigen, whereinthe peptide is 20 or fewer amino acids in length, wherein the peptide islinked to the therapeutically active drug to inhibit the therapeuticactivity of the drug, and wherein the therapeutically active drug iscleaved from the peptide upon proteolysis by an enzyme having aproteolytic activity of prostate specific membrane antigen.
 2. Thecomposition of claim 1, wherein the peptide has the general structure X₁. . . X_(n), wherein n is from 2 to 20, and wherein X₁ is an amino acidselected from the group consisting of aspartic acid and glutamic acid,X₂-X_(n−1) are amino acids selected from the group consisting ofaspartic acid, glutamic acid, asparagine and glutamine, and X_(n) is anamino acid selected from the group consisting of aspartic acid, glutamicacid, asparagine and glutamine.
 3. The composition of claims 1 or 2,wherein the peptide is linked directly to an amino group or to acarboxyl group of the therapeutic drug.
 4. The composition of claims1-3, wherein the therapeutically active drug is selected from the groupof primary amine containing thapsigargins or thapsigargin derivatives.5. The composition of claims 1-3, wherein the therapeutically activedrug is an antifolate.
 6. The composition of claim 5, wherein thetherapeutically active drug is selected from the group consisting ofmethotrexate, ralitrexed, edatrexate, lometrexol, and pemetrexed.
 7. Thecomposition of claim 5, wherein the therapeutically active drug isselected from the group consisting of amino acid-containing analogs oftrimetrexate.
 8. The composition of claims 1-3, wherein thetherapeutically active drug is an anthracycline antibiotic.
 9. Thecomposition of claim 8, wherein the therapeutically active drug isselected from the group consisting of doxorubicin, daunorubicin,epirubicin and idarubicin.
 10. The composition of claims 1-3, whereinthe therapeutically active drug is a taxane.
 11. The composition ofclaim 10, wherein the therapeutically active drug is selected from thegroup consisting of taxotere and taxol.
 12. The composition of claim 2,wherein n is 2, X₁ is aspartic acid and X₂ is glutamic acid.
 13. Thecomposition of claim 2, wherein n is 5, X₁ is aspartic acid, X₂ isglutamic acid, X₃ is glutamic acid, X₄ is aspartic acid, X₅ is glutamicacid, and wherein X₂ is linked to the side chain of X₃, and X₃ is linkedto the side chain of X₄.
 14. A method of producing a prodrug, the methodcomprising the step of linking a therapeutically active drug and apeptide comprising an amino acid sequence having a cleavage sitespecific for an enzyme having a proteolytic activity of prostatespecific membrane antigen, wherein the peptide is 20 or fewer aminoacids in length, wherein the peptide is linked to the therapeuticallyactive drug to inhibit the therapeutic activity of the drug, and whereinthe therapeutically active drug is cleaved from the peptide uponproteolysis by an enzyme having a proteolytic activity of prostatespecific membrane antigen.
 15. The method of claim 14, wherein thetherapeutically active drug has a primary amine.
 16. The method of claim14, wherein the therapeutically active drug has a carboxyl group.
 17. Amethod of treating a PSMA-producing cell proliferative disorder, themethod comprising administering the composition of claims 1 and 2 in atherapeutically effective amount to a subject having the cellproliferative disorder.
 18. The method of claim 17, wherein the disorderis benign.
 19. The method of claim 17, wherein the disorder ismalignant.
 20. The method of claim 19, wherein the malignant disorder isprostate cancer.
 21. The method of claim 19, wherein the malignantdisorder is breast cancer, pancreatic cancer, brain cancer, melanoma,lung cancer, testicular cancer, or sarcoma.
 22. A method of detectingprostate specific membrane antigen-producing tissue, the methodcomprising: contacting the tissue with a composition comprising adetectably labeled peptide comprising an amino acid sequence having acleavage site specific for an enzyme having a proteolytic activity ofprostate specific membrane antigen, wherein the peptide is 20 or feweramino acids in length for a period of time sufficient to allow cleavageof the peptide; and detecting the detectable label.
 23. A method ofselecting a prostate specific membrane antigen-activatable prodrugwherein the prodrug is substantially specific for target tissuecomprising prostate specific membrane antigen-producing cells, themethod comprising: a) linking a peptide comprising an amino acidsequence having a cleavage site specific for an enzyme having aproteolytic activity of prostate specific membrane antigen, wherein thepeptide is 20 or fewer amino acids in length to a therapeutic drug toproduce a peptide-drug composition; b) contacting the composition withcells of the target tissue; c) contacting the composition with cells ofa non-target tissue; and d) selecting compositions that aresubstantially toxic towards target tissue cells, but which are notsubstantially toxic towards non-target tissue cells.
 24. A method ofdetermining the activity of prostate specific membrane antigen in asample containing PSMA, the method comprising: a) contacting the samplewith a composition comprising a detectably labeled peptide comprising anamino acid sequence having a cleavage site specific for an enzyme havinga proteolytic activity of prostate specific membrane antigen, whereinthe peptide is 20 or fewer amino acids in length for a period of timesufficient to allow cleavage of the peptide; b) detecting the detectablelabel to yield a detection level; c) comparing the detection level witha detection level obtained from contacting the detectably labeledpeptide with a standard prostate specific membrane antigen sample.
 25. Amethod of imaging prostate specific membrane antigen-producing tissue,the method comprising: a) administering a peptide linked to a lipophilicimaging label to a subject having or suspected of having a prostatespecific membrane antigen producing-associated cell-proliferativedisorder; b) allowing a sufficient period of time to pass to allowcleavage of the peptide by prostate specific membrane antigen and toallow clearance of uncleaved peptide from the subject to provide areliable imaging of the imaging label; and c) imaging the subject.